Synaptobrevin-2/VAMP-2 (syb2) is an abundant synaptic vesicle protein essential for normal synaptic transmission in the brain. Syb2's interaction with the plasma membrane proteins, syntaxin 1 and SNAP-25, is critical for synaptic vesicle fusion and neurotransmitter release. These proteins are collectively called SNAREs (acronym for soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and belong to a family of proteins that mediate vesicle trafficking and fusion in the secretory pathway in eukaryotes. Despite extensive progress in the characterization of molecular interactions among SNAREs and their role in fusion, their precise role in synaptic vesicle trafficking events after fusion remains elusive. Rapid coupling of vesicle fusion and retrieval during neurotransmission have led us to hypothesize that SNARE proteins that drive rapid Ca2+ dependent fusion may also be responsible for ensuring rapid synaptic vesicle retrieval. Indeed, our studies in the previous grant period have revealed an essential role for syb2 in rapid synaptic vesicle endocytosis. Moreover, our initial results suggest that this function of syb2 may not be shared by SNAP-25. This observation suggests a specific role for syb2 in ensuring faithful coupling between exocytosis and endocytosis. In the next award period, we aim to investigate the role of syb2 and related v-SNAREs in exo-endocytic coupling, synaptic vesicle trafficking after endocytosis as well as fusion pore regulation using a powerful combination of fluorescence imaging, electrophysiology and electron microscopy. For this purpose, we propose three aims. In the first aim, we will define the role of v-SNAREs in coupling exocytosis and endocytosis via monitoring trafficking of fluorescently-tagged v-SNAREs synaptic vesicle proteins. In the second aim, we will determine the function of v-SNAREs in postendocytic trafficking of synaptic vesicles by detecting uptake and release fluorescent probes, monitoring neurotransmitter release and electron microscopy. Lastly, we will determine the impact of v-SNAREs on unitary neurotransmission and glutamate release kinetics using optical and electrophysiological measures. Collectively, these experiments will elucidate the degree of overlap between the fusion machinery and endocytic machinery in central synapses and the role of SNAREs in directing synaptic vesicle trajectories during retrieval, vesicle reuse as well as neurotransmitter release. Information attained from these studies will provide new insight to the synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia. PUBLIC HEALTH RELEVANCE: The experiments proposed for this project present a systematic and comprehensive effort to address the role of key SNARE molecules in the regulation of synaptic vesicle fusion, retrieval and recycling. Currently, a thorough analysis of the role of SNAREs in synaptic vesicle trafficking beyond vesicle fusion is lacking. In this project, we aim to establish the basic principles of SNARE-dependent regulation of synaptic vesicle trafficking in mammalian central synapses. Information attained from these studies will provide new insight to the molecular synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia.